Cell Differentiation and Development, 32 (1990) 247-254 247 © 1990 Elsevier Scientific Publishers Ireland, Ltd. 0922-3371/90/$03.50

CELDIF 99924

The urokinase receptor and regulation of cell surface plasminogen activation

Francesco Blasi 1, Niels Behrendt 2, M. Vittoria Cubellis 1, Vincent Ellis 2, Leif R. Lund 2 M. Teresa Masucci 1, Lisbeth B. Meller 1, David P. Olson 1, Nina Pedersen 1, Michael Ploug 2,

Ebbe Ronne 2 and Keld Dano 2

/ Institute of Microbiology, University of Copenhagen, Copenhagen, Denmark and 2 Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark

Urokinase-type plasminogen activator; Urokinase receptor; Metastasis

Urokinase-type plasminogen activator (u-PA) is a key enzyme involved in migration and invasive- ness of cells, both in cancer and in several normal physiological processes (Reich, 1978; Dana et al., 1985; Saksela and Rifkin, 1988; Blasi and Verde, 1990). The application of modem biochemical and molecular biological techniques has identified some of the steps at which u-PA activity is regu- lated.

A large number of studies have dealt with the biological roles of u-PA catalyzed plasminogen activation. These can be summarized as follows: (1) u-PA-dependent proteolysis can take place on cells with surface-bound reactants; physiologi- cally, this may well be the most relevant form of plasminogen activation; (2) the plasminogen activating system and its regulation are complex and several molecules are known to be involved (activators, substrate, inhibitors, receptors), al- though other, as yet unidentified, components are also implicated and (3) on a biochemical basis the u-PA system exploited by cancer cells appears to be qualitatively identical to that used in normal

Correspondence address: F. Blasi, Institute of Microbiology, University of Copenhagen, Oster Farimagsgade 2A, 1353 Copenhagen K., Denmark.

physiological invasive processes. Possibly, the fundamental difference between cancer cells and normal cells in this respect lies in the regulation of the system.

A connection between u-PA and metastasis has been found in a model system by Ossowski and Reich (1983), who showed that human HEp3 cells inoculated onto the chorio-aklantoic membrane of the chicken embryo metastasize into the lung of the embryo and that this process can be blocked by specific anticatalytic anti-human u-PA antibod- ies. In other model systems, only some of the aspects of the metastatic phenotype can be in- vestigated. For example, the lung-colonizing abil- ity of mouse B16F10 melanoma cells inoculated in the tail vein of syngeneic mice can be strongly limited by pre-treatment of the cells with specific anti-u-PA antibodies (Heating et al., 1988). Over- expression of u-PA in ras-transformed NIH 3T3 cells increases their capacity to colonize the lung after tail-vein injection (Axelrod et al., 1989). Other experiments have shown an involvement of tumor cell u-PA in model systems measuring degradation of the extracellular matrix and the invasion of the basement membrane in vitro (Bergman et al., 1986; Mignatti et al., 1986; Cajot et al., 1990).

A variety of non-malignant physiological processes that involve tissue degradation and cell

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migration also appear to involve u-PA activity: trophoblast invasion (Strickland et al., 1976; Sap- pino et al., 1989), postlactational mammary gland involution (Ossowski et al., 1979), wound healing (Gr~ndahl-Hansen et al., 1988), spermatogenesis (Vihko et al., 1988) and prostate involution (Andreasen et al., 1990) are typical examples.

Additional relevant findings of recent years in- clude the demonstration of a strong enhancement of u-PA catalyzed plasminogen activation when both pro-u-PA/u-PA and plasminogen are bound to cell surfaces, the identification and purification of a specific receptor for u-PA, and the cloning of a complete cDNA for this receptor.

The urokinase receptor (u-PAR)

u-PAR was detected by a specific and saturable binding of u-PA to monocytes and monocyte-like cells (VassaUi et al., 1985; Stoppelli et al., 1985) and has been found on the surface of many types of cultured cells, of both normal and neoplastic origin (Bajpai and Baker, 1985; Plow et al., 1986; Stoppelli et al., 1986; Boyd et al., 1988; Nielsen et al., 1988; for reviews, see Blasi et al., 1987; Blasi, 1988; Dano et al., 1990). The receptor binds both u-PA and its proenzyme pro-u-PA (Cubellis et al., 1986) with a high affinity ( g o = 1 0 - 9 - - 10-riM), which varies between cell types (Blasi, 1988). Pro- u-PA can be converted to active u-PA when bound to the receptor and receptor-bound u-PA can in turn activate plasminogen (Cubellis et al., 1986; Stephens et al., 1989; Ellis et al., 1989). Concom- itant binding of pro-u-PA to u-PAR and of plas- minogen to as yet unidentified binding sites at the cell surface (Plow et al., 1986; Hajjar et al., 1986; Burtin and Fondaneche, 1988) strongly enhances plasmin formation (Ellis et al., 1989; Stephens et al., 1989). The mechanism underlying this en- hancement is not completely understood, although the primary cause appears to be an increased activation of pro-u-PA by cell-associated plasmin (Ellis et al., 1989). In some cell types u-PAR focalize u-PA to cell-cell and cell-substratum con- tact sites (PiSllanen et al., 1987, 1988; H6bert and Baker, 1988).

Purification and characterization of u-PAR

u-PAR has been purified from phorbol myri- state acetate (PMA)-stimulated U937 cells by tem- perature-dependent phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fiuorophosphate-treated u-PA (Nielsen et al., 1988; Behrendt et al., 1990). Using the covalent cross-linker N,N'-disuc- cinimidyl suberate, the purified protein can be cross-linked to u-PA; identical electrophoretic mo- bilities were observed for cross-linked conjugates formed with either the purified protein or the u-PA binding component on the surface of intact U937 ceils. Antibodies raised against the purified protein inhibit cellular binding of the amino- terminal fragment of u-PA (ATF) which contains the receptor-binding determinant.

The purified protein behaves as a single poly- peptide with apparent M r 55-60,000 during SDS-PAGE. It is highly glycosylated; the deglyco- sylated polypeptide chain migrated corresponding to only M r 35,000. The fully processed protein contains siaiic acid and N-acetyl-D-glucosamine, indicative of N-linked carbohydrate. No N-acetyl- D-galactosamine was demonstrated, thus exclud- ing the presence of at least one type of O-linked carbohydrate. Glycosylation is responsible for a substantial M r heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines.

Cloning and expression of cDNA for u-PAR

An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified u-PAR was used to screen a cDNA library made from SV40 transformed human fibroblasts. A 1.4 kb cDNA clone coding for the entire human u-PAR was isolated (Roldan et al., 1990). The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. Expression of the u-PAR cDNA in mouse cells confirmed that the clone is complete and expresses a functional u-PA binding protein, located at the cell surface and with properties similar to the human u-PAR.

Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human u-PA, which in turn can activate plasminogen. The mature human re- ceptor protein expressed in mouse cells can be covalently cross-linked to ATF, and the M r de- duced from SDS-PAGE of the formed conjugate is 50-55,000, in accordance with that of the natu- rally occurring, highly glycosylated human u-PAR. The M r = 35,000, calculated on the basis of the cDNA sequence, agrees well with that of the de- glycosylated receptor.

Regulation of u-PAR synthesis by PMA

In U937 cells, PMA causes an early increase in u-PAR mRNA level, which reaches a maximal 50-fold enhancement after 24 h of treatmemt (Lund et al., 1991). Half maximal stimulation oc- curs at = 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promo- ters. The protein synthesis inhibitor cycloheximide (10 #g/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments show a time-dependent increase in the u-PAR gene tran- scription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand.

u-PAR has a glycosyl-phosphatidyl inositol mem- brane anchor

Recently, human u-PAR has been shown by several independent criteria to belong to a group of integral membrane proteins, which are anchored to the plasma membrane exclusively by a COOH- terminal glycosyl-phosphatidylinositol moiety (Ploug et al., 1991). The following properties of u-PAR are a consequence of this unique mode of membrane anchorage by glycolipids: (1) analysis of ortho-phtalic dialdehyde reactive components in the hydrolysates of u-PAR, purified by affinity chromatography and Tricine-SDS-PAGE, re-

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vealed the presence of 2 - 3 mol ethanolamine/ mol protein; (2) membrane bound u-PAR was efficiently released from the surface of human U937 cells by catalytic amounts of purified bacterial phosphatidylinositol-specific phospholi- pase C (PI-PLC). This soluble form of u-PAR retained the binding specificity towards both u-PA and its amino terminal fragment (ATF) holding the receptor-binding domain; (3) treatment of purified u-PAR with PI-PLC or mild alkali alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis; (4) biosynthetic labeling of u-PAR was obtained with both [3H]ethanolamine and myo-[3H]inositol and finally (5) comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a COOH terminus polypeptide is excised from the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane an- chor (Ferguson et al., 1988).

Interaction of receptor-bound u-PA with the inhibi- tors PAI-1 and PAI-2

In addition to its interaction with u-PAR, u-PA interacts with its specific inhibitors, plasminogen activator inhibitor type 1 and 2 (PAI-1 and PAI-2). The interactions of u-PA with u-PAR and the inhibitors, respectively, are mediated by two func- tionally independent domains of the molecule: the catalytic (within the heavy polypeptide chain) and the growth factor-like domain (in the light chain). It was found that PAI-1 can also bind to receptor-bound uPA (Cubellis et al., 1989). Bind- ing of 125I-labeled ATF to human U937 mono- cyte-like cells can be competed for by u-PA/PAI-1 complexes, but not by PAI-1 alone. Preformed, 125I-labeled u-PA/PAI-1 complexes can bind to the cell surface with the same binding specificity as u-PA, i.e. requiring the presence of the ATF part of the enzyme.

A kinetic analysis showed that u-PA, specifi- cally bound to u-PAR on human U937 cells, is inhibited by PAI-1 with an association rate con-

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stant of 4.5 × 10 6 M -1 S -1, which is only 40% lower than that obtained for u-PA in solution (7.9 × 106 M -l s -1) (Ellis et al., 1990). The in- hibition of u-PA by PAI-2 is decreased to a simi- lar extent by receptor binding, falling from 5.3 x 105 to 3.3 × 105 M -l s -k Stimulation of U937 cells with phorbol 12-myristate 13-acetate is accompanied by some further reduction in the rate constants for inhibition of receptor-bound u-PA (to 1.7 × 106 and 1.1 × 105 M -l s -x for PAI-1 and PAI-2, respectively. These constants, although somewhat lower than those for u-PA in solution, still represent a rather rapid inhibition of the enzyme, and demonstrate that receptor-bound u-PA remains available for efficient inhibition by PAIs, which may therefore play a major role in controlling cell-surface plasminogen activation.

Internalization of u-PAR bound complexes be- tween u-PA and PAl-1

The receptor for u-PA has been shown not to internalize its ligand (Vassalli et al., 1985; Stop- pelli et al., 1985, 1986); u-PA/PAI-1 complexes bound to the receptor are, however, internalized and degraded (Cubellis et al., 1990). U937 cells were incubated at 4°C with labeled u-PA/PAI-1 (and other ligands), the temperature then raised to 37 °C and the fate of the ligand followed for 3 h. The u-PA/PAI-1 complex was internalized into the cells (i.e. could not be dissociated by acid treatment) and thereafter degraded (i.e. appeared in the supernatant in a non TCA-precipitable form). Other ligands (uncomplexed u-PA, ATF and DFP-treated u-PA) were neither internalized nor degraded. The degradation of the u-PA/PAI-1 complex is preceded by internalization and is in- hibited by chloroquine, an inhibitor of lysosomal protein degradation.

Regulation of cell surface plasminogen activation

Under physiological conditions, u-PA catalyzed cell surface plasminogen activation appears to re- quire in addition to u-PA also the presence of u-PAR and the absence of PAI-1 and PAI-2. The

synthesis of each of these four components is regulated by a variety of hormones, growth factors and cytokines. The pattern of regulation varies among cell types. If, for example, a certain growth factor induces u-PA synthesis in one cell type, it may inhibit it in another and leave it unchanged in most cell types. In a certain cell type the regulation may be concerted so that a certain factor for example may decrease u-PA and u-PAR and increase PAI-1 and PAI-2. In most cases such a concerted action, however, is absent (see Dano et al., 1985, 1988; Blasi and Verde, 1990).

This provides a versatile system for regulation of cell surface plasminogen activation that allows regulatory factors in the microenvironment to turn on plasminogen activation on one cell type but not on others. Such a system may form the basis for a strict regulation of plasminogen activation during non-malignant physiological processes such as trophoblast invasion, wound healing and pros- tate involution, while defects in this regulatory system may lead to the apparently uncontrolled plasminogen activation in cancer.

The recent findings on the u-PA receptor indi- cate that this molecule plays a central role in cell surface plasminogen activation and that the regu- lation of this process may be much more com- plicated and diverse than outlined above.

Thus, not only the synthesis of u-PAR but also its affinity for u-PA is subject to regulation as shown by the effect of prolonged treatment of human U937 and HeLa cells with phorbol esters and epidermal growth factor, which decrease this affinity dramatically (Picone et al., 1989; Es- treicher et al., 1989). Nothing is known as yet of the mechanism of this regulation which may in- volve the receptor directly or take place through so far undetected regulatory molecules interacting with u-PAR.

Another level of regulation of cell surface plasminogen activation may be the activation of pro-u-PA. The conversion of receptor-bound pro- u-PA to active two-chain u-PA takes place at a much higher rate than in solution (Ellis et al., 1989); this reaction appears to be catalyzed by cell-surface associated plasmin, and causes a strong amplification of the overall reaction as soon as it has been initiated. However, the mechanism of the

initiation itself has not yet been clarified. The overall amplification of plasminogen activation also appears to be favored by a degree of protec- tion of the system from serum protease inhibitors (Stephens et al., 1989), notably a2-antiplasmin (Plow et al., 1986). The detailed mechanisms un- derlying these cell-associated proteolytic reactions have not been deduced, and are complicated by the difficulty in distinguishing between direct re- actions among surface-located components in an organized manner and effects caused by locally increased solution concentrations in the pericellu- lar microenvironment due to a high density of the reactants reversibly bound to the cell surface. However, recent kinetic evidence strongly suggests that this system of plasminogen activation can occur entirely in association with the cell-surface (V. Ellis and K. Dane, unpublished results).

The fact that PAI-1 can bind, inhibit and cause the internalization and degradation of receptor- bound u-PA offers further possibilities for regu- lation. In cultures of HT1080 fibrosarcoma cells, PAI-1 and u-PA do not co-localize, receptor-bound u-PA being located at cell-cell and focal cell-sub- stratum contact sites, while PAI-1 is associated with the extracellular matrix, presumably bound to vitronectin (PiSllLnen et al., 1987, 1988; H6bert and Baker, 1988; Salonen et al., 1989). Possibly, migration of cells puts the receptor-bound u-PA in-contact with the extracellular matrix-bound PAI-1. Thus cell migration might in itself regulate surface proteolysis; if internalization and degrada- tion of receptor-bound u-PA by PAI-1 go through an endocytotic step, then migrating cells are endo- wed with the properties of carrying out a complete plasminogen-activation sequence, starting with the synthesis and secretion of inactive pro-u-PA, fol- lowed by binding to u-PAR, activation, produc- tion of surface plasmin, inhibition by PAI-1 and internalization and degradation of the receptor- bound u-PA/PAI-1 complex.

Still another form of regulation can be en- visaged, u-PA can be cleaved at residue 135 into two distinct functionally active domains: the carboxy terminal domain endowed with enzyme activity and inhibitor-binding (but not receptor- binding) capacity, and the amino terminal domain (ATF) which has no enzyme activity, but can bind

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the receptor (Stoppelli et al., 1985). Since the half life of receptor bound ATF is quite long (Cubellis et al., 1990), the cleavage of u-PA at residue 135 might result in the occupation of the receptor with an inactive, yet stable ligand, decreasing the actual as well as the potential surface-bound activity. The released carboxy terminal moiety of u-PA, although enzymatically active, will probably be inhibited by the specific inhibitors.

The finding that u-PAR is bound to cell surfaces by a glycosyl-phosphatidyl inositol membrane anchor and can be released by a bacterial phos- phatidyl inositol-specific phospholipase C suggests the possibility of still another type of regulatory mechanism. Even without PI-PLC treatment there are small but definite amounts of water soluble u-PAR present in conditioned medium from U937 cells (M. Ploug et al., 1991). This may be the result of the action of putative human phospholipases, and such phospholipases may have a regulatory function. In this context it is noteworthy that in contrast to the enhanced plasminogen activation observed after concomitant binding of u-PA (through u-PAR) and plasminogen to cell surfaces, purified u-PAR has some, although not a com- plete, inhibitory effect on u-PA catalyzed plas- minogen activation in solution (V. Ellis and N. Behrendt, unpublished observations).

u-PAR may thus participate in the regulation of the u-PA pathway of plasminogen activation not only by the overall enhancement of plasmin formation due to the concomitant binding of pro- u-PA and plasminogen to cell surfaces, but also in other ways to give a more finely tuned temporal and spatial regulation of this process.

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